Existing thrombolytic drugs, used in the treatment of thromboembolic diseases, have limited effectiveness and also carry the risk of rethrombosis and hemorrhagic complications. Clinical experience with plasminogen activators has highlighted problems with both efficacy and side effects, particularly hemorrhage [Rao et al. J Amer Coll Cardiol. 11: 1-11 (1988); Fennerty et al. Chest. 95: 88S-97S (1989)]. Because both endpoints are dose-related, the efficacy of therapeutic thrombolysis has always been handicapped by its side effects.
Currently most therapeutic thrombolysis is performed using tissue plasminogen activator (tPA) and its derivatives. TPA can have hemorrhagic side effects. For example, tissue plasminogen activator (tPA) at a dose of 150 mg has been shown to induce superior coronary thrombolysis, but has been accompanied by an unacceptable incidence of intracranial hemorrhage, obliging the adoption of a less effective dose of 100 mg [Braunwald et al. J Amer Coll Cardiol. 9: 467 (1987); Grossbard. J Amer Coll Cardiol. 9:467 (1987)].
Similarly, the other natural plasminogen activator, single-chain urokinase plasminogen activator (prouPA), a proenzyme, requires high infusion rates for effective coronary thrombolysis which causes plasminemia and results in conversion of single to two-chain uPA (tcuPA) and bleeding [Meyer et al. Lancet 1:863-868 (1989)].
The bleeding complications of therapeutic thrombolysis have been ascribed to the direct lysis of hemostatic fibrin at a vascular injury site and to the hemorrhagic diathesis caused by non-specific plasmin generation resulting in fibrinogenolysis, degradation of fibrinogen and of clotting factors V and VIII. Fibrinogen is the principal protein constituent of a fibrin clot; clotting factor V is a cofactor in the coagulation system, the lack of which causes a predisposition for hemorrhage; and clotting factor VIII is an essential clotting factor, the lack of which causes Hemophilia A.
Both tPA and prouPA are fibrin-specific in that they preferentially activate plasminogen bound to fibrin over free plasminogen. At physiological concentrations, plasminogen activation by tPA and prouPA is fibrin-dependent and confined to the clot environment by plasma inhibitors. But, at therapeutic concentrations, the fibrin selectivity of tPA and prouPA is compromised, largely due to the fact that these concentrations are in excess of inhibitors, particularly plasminogen activator inhibitor-1 (PAI-1), their principal plasma inhibitor. The intrinsic activity of prouPA at therapeutic concentrations was sufficient to activate plasma plasminogen, which converted single-chain prouPA to two-chain uPA (tcuPA). Since tcuPA is a non-specific plasminogen activator, the fibrin-specificity of prouPA is lost. Thus prouPA's specificity depends on its plasma stability which allows tcuPA and plasmin generation to be confined to the fibrin clot [Pannell and Gurewich, Blood, 67: 1215-1223 (1986)]. The systemic activation of plasma plasminogen results in the generation of systemic tcuPA and undermines the therapeutic use of prouPA.
At therapeutic concentrations, prouPA is especially vulnerable to non-specific plasmin generation since this results in loss of its proenzyme configuration due to its conversion to tcuPA, a non-specific activator, which, being an enzyme, then amplifies systemic plasmin generation several hundred fold.
This cycle of reactions is initiated by the relatively high intrinsic activity of prouPA which at therapeutic concentrations triggers plasminogen activation. Therefore, a prouPA mutation (M5) with a lower intrinsic catalytic activity was developed. A five-fold reduction in intrinsic activity was achieved by a site-directed single residue exchange on a flexible loop in the catalytic domain (Lys300→His) of prouPA [Liu, et al. Biochemistry 35: 14070-14076 (1996)]. This produced a corresponding degree of improvement in plasma stability or inertness at therapeutic concentrations. Unexpectedly, after activation to two-chain M5 (tcM5), the mutant had a two-chain activity almost twice that of tcuPA [Sun et al., J Biol. Chem. 272: 23818-23823 (1997)], consistent with their two-chain active catalytic sites also being functionally distinct. U.S. Pat. No. 5,472,692 describes prouPA mutants and the disclosure is incorporated herein by reference.
M5 induced efficient, fibrin-specific clot lysis in a plasma milieu in vitro and in dogs with venous thromboemboli in which M5 was associated with no more bleeding than placebo [Liu et al. Circ Res. 90: 757-763 (2002)] In a second animal study of M5, a more challenging arterial thrombus was selected and M5 was administered by a bolus/infusion administration modeled on the clinical administration of prouPA or tPA. Because blood loss from injury sites was the side effect of most concern, a more quantitative measure of blood loss was used. Furthermore, the plasma inhibition of tcM5 was studied and found to be related to a plasma inhibitor novel for tcuPA. M5 and tPA induced comparably effective lysis, but blood loss from fresh hemostatic sites was ten-fold higher with tPA, suggesting that M5 spared hemostatic fibrin at doses which lyse intravascular clots [Pannell et al. Blood. 69: 22-26 (1987)]. A difference in the lytic sensitivities of hemostatic versus intravascular fibrin to M5 was related to differences in the mechanisms of fibrin-dependent plasminogen activation by the two activators [Gurewich et al. J Thromb Haemost. 4: 1559-65 (2006)]. Specifically, M5 selectively activated plasminogen on partially degraded (fibrin fragment E) and not on intact fibrin, whereas tPA targeted plasminogen on intact fibrin (fibrin fragment D) [Liu et al. J Clin Invest. 88: 2012-2017 (1991)], which corresponds to hemostatic fibrin. However, a novel, additional explanation for the low bleeding rate with M5 also came to light in this study [Gurewich et al. J Thromb Haemost. 4: 1559-65 (2006)].
Zymography of plasma samples from dogs in the dose-finding phase in which higher infusion rates of M5 were used, and where non-specific activation occurred, showed an unusual inhibitor complex with tcM5. This complex was also seen when tcM5 (but not M5) was incubated in vitro in dog or human plasma. The inhibitor was identified as C1-inhibitor based on its co-migration with a complex formed with purified C1-inhibitor and Western blotting with specific antibodies. It was postulated that endogenous C1-inhibitor helped confine tcM5 activity to the fibrin-clot environment, thereby limiting non-specific plasminogen activation and sparing hemostatic fibrin in these dogs [Gurewich et al. (2006), supra]. In the present study, C1-inhibitor inhibition of tcM5 was further investigated and its effect on fibrin-specific and non-specific plasminogen activation by M5 was characterized in vitro.